OLIGODENDROGLIAL APOPTOSIS OCCURS ALONG DEGENERATING AXONS AND IS ASSOCIATED WITH FAS AND p75 EXPRESSION FOLLOWING SPINAL CORD INJURY IN THE RAT S. CASHA, W. R. YU and M. G. FEHLINGS* Divisions of Neurosurgery and Cell and Molecular Biology, Toronto Western Research, The University Health Network, Toronto Western Hospital, University of Toronto, Toronto, Ontario, Canada Abstract ÐApoptosis or programmed cell death has been reported after CNS trauma. However, the signi®cance of this mechanism in the pathophysiology of spinal cord injury, in particular at the cervical level, requires further investigation. In the present study, we used the extradural clip compression model in the rat to examine the cellular distribution of apoptosis following cervical spinal cord injury, the relationship between glial apoptosis and post-traumatic axonal degeneration and the possible role of apo[apoptosis]-1, CD95 (FAS) and p75 in initiating post-traumatic glial apoptosis. In situ terminal- deoxy-transferase mediated dUTP nick end labeling revealed apoptotic cells, largely oligodendrocytes as identi®ed by cell speci®c markers, in grey and white matter following spinal cord injury. Apoptotic cell death was con®rmed using electron microscopy and by the demonstration of DNA laddering on agarose gel electrophoresis. b-Amyloid precursor protein was used as a molecular marker of axonal degeneration on western blots and immunohistochemistry. Degeneration of axons was temporally and spatially co-localized with glial apoptosis. FAS and p75 protein expression was seen in astrocytes, oligo- dendrocytes and microglia, and was also seen in some apoptotic glia after cord injury. Both FAS and p75 increased in expression in a temporal course, which mirrored the development of cellular apoptosis. The downstream caspases 3 and 8, which are linked to FAS and p75, demonstrated activation at times of maximal apoptosis, while FLIP-L an inhibitor of caspase 8, decreased at times of maximal apoptosis. We conclude that axonal degeneration after traumatic spinal cord injury is associated with glial, in particular oligo- dendroglial, apoptosis. Activation of the FAS and p75 death receptor pathways may be involved in initiating this process. q 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: programmed cell death, neurotrauma, glia, caspases, death receptors, b-amyloid precursor. Apoptosis is an active process involving activation of dedicated cellular suicide machinery (programmed cell death). It is typi®ed by cytoplasmic condensation, endonuclease activation, chromatin condensation and DNA fragmentation, followed by phagocytosis. 13,32,54 Necrosis does not necessarily involve the activation of an intracellular program. It is characterized by cell and organelle swelling with subsequent loss of membrane integrity, resulting in spilling of the intracellular contents, eliciting an in¯ammatory response. 54 While, necrotic cell death has been long recognized to occur following neural trauma, recent evidence has implicated apoptotic mechanisms as well. 16,39,41,59 The pathophysiology of spinal cord injury (SCI) involves an initial primary or mechanical insult followed by a series of secondary events including ischemia, ionic ¯uxes, free radical production and lipid peroxidation, which result in further cell death. 1,2,69 Several features of SCI suggest that apoptosis plays a signi®cant role in the pathophysiology of this condition. Increases in intra- cellular Ca 21 occur after SCI and are important in post- traumatic cell death. 72 Ca 21 -dependent breakdown of DNA and protein also occurs in apoptosis. 37 Hypoxia and free radical formation follow SCI, and are processes known to trigger p53 mediated DNA repair and apopto- sis. 6,28 Glutamate excitotoxicity has been established as a key event following neural trauma and has also been linked to apoptosis. 2,17,30,38 A number of studies have now provided evidence of Oligodendroglial apoptosis after cervical cord injury 203 203 Neuroscience Vol. 103, No. 1, pp. 203±218, 2001 q 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/01 $20.00+0.00 PII: S0306-4522(00)00538-8 Pergamon www.elsevier.com/locate/neuroscience *To whom correspondence should be addressed. Tel.: 11-416- 603- 5229; fax: 11-416-603-5745. E-mail address: michael@uhnres.utoronto.ca (M. G. Fehlings). Abbreviations: AEC, aminoethyl carbazole; AMPA, a-amino-3- hydroxy-5-methyl-4-isoxaline proponic acid; ASK1, apoptosis signal-regulating kinase 1; bAPP, b-amyloid precursor protein; Caspase, cysteine protease which cleaves at an aspartate residue; CNP, 2',3'-cyclic nucleotide 3'-phosphodiesterase; DAXX, FAS death domain-associated protein; EDTA, ethylenediaminetetra- acetate; FAS, Fas receptor (aka Apo[apoptosis]-1, CD95); FADD, FAS-associated death domain; FAS-L, FAS ligand; FITC, ¯uorescein isothiocyanate; FLICE, caspase 8/FADD-like IL-1 beta-converting enzyme; FLIP, FLICE (caspase 8) inhibitory protein; GFAP, glial ®brillary acidic protein; GGF, glial growth factor; HRP, horseradish peroxidase; MAPK, mitogen-activated protein kinase; MK801, (5R, 105)-(1)-5-methyl-10,11-dihydro- SH-dibenzo[a,b]-cyclohepten-5,10-imine; NF200, neuro®lament 200; NGF, nerve growth factor; NMDA, N-methyl-d-aspartate; p75, low-af®nity neurotropin receptor; PBS, phosphate-buffered saline; RNAase, Ribonuclease; SCI, spinal cord injury; SDS± PAGE, sodium dodecylsulphate±polyacrylamide gel electrophor- esis; T cells, thymus derived lymphocyte; TNFR1, tumor necrosis factor receptor 1 (a.k.a. p55, CD120a); TRIS, Tris(hydroxymethy- l)aminomethane phosphate; TrkA, tyrosine kinase receptor A; TTBS, 0.1% Tween 20 in Tris-buffered saline; TUNEL, in situ terminal-deoxy-transferase mediated dUTP nick end labeling.